Search

CN-122011193-A - Anti-MDM 2 single-chain antibody and application thereof

CN122011193ACN 122011193 ACN122011193 ACN 122011193ACN-122011193-A

Abstract

The invention discloses an anti-MDM 2 single-chain antibody and application thereof, in particular to an anti-MDM 2 single-chain antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs, H-CDR1, H-CDR2 and H-CDR3, the light chain variable region comprises 3 CDRs, L-CDR1, L-CDR2 and L-CDR3, and the amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are as follows Any one of the CDR1, CDR2 and CDR3 of the heavy chain variable region, wherein the amino acid sequences of the L-CDR1, L-CDR2 and L-CDR3 are as follows CDR1, CDR2, CDR3 of any one of the light chain variable regions shown.

Inventors

  • LI JINGHONG
  • LI YUE
  • WANG LINGXIAO
  • ZHAO XUAN

Assignees

  • 北京生命科技研究院有限公司

Dates

Publication Date
20260512
Application Date
20260108

Claims (10)

  1. 1. An antibody or antigen-binding fragment thereof which specifically binds to MDM2 protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising 3 CDRs, H-CDR1, H-CDR2 and H-CDR3 and a light chain variable region comprising 3 CDRs, L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are as follows Any one of the CDR1, CDR2 and CDR3 of the heavy chain variable region, wherein the amino acid sequences of the L-CDR1, L-CDR2 and L-CDR3 are as follows CDR1, CDR2, CDR3 of any one of the light chain variable regions shown.
  2. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the CDRs of the light chain variable region and the heavy chain variable region are selected from any one of the following: (a) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 7, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 13; (b) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 8, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 14; (c) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 9, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 15; (d) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 10, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 16; (e) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 11, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 17; (f) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 12, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region shown as SEQ ID No. 18.
  3. 3. A nucleic acid molecule or a vector comprising said nucleic acid molecule, wherein said nucleic acid molecule encodes the antibody or antigen-binding fragment thereof of claim 1 or 2.
  4. 4. A cell comprising the nucleic acid molecule or vector of claim 3.
  5. 5. A fusion protein comprising the antibody or antigen-binding fragment thereof of claim 1 or 2.
  6. 6. A reagent or kit comprising the antibody or antigen-binding fragment thereof of claim 1 or 2.
  7. 7. A pharmaceutical formulation comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, and a pharmaceutical carrier.
  8. 8. A method for detecting MDM2 protein for non-diagnostic purposes, characterized in that the antibody or antigen binding fragment thereof according to claim 1 or 2 is used for co-incubation with a test sample.
  9. 9. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2, or the pharmaceutical formulation of claim 7, in any of the following: (1) Use in the manufacture of a medicament for the prevention or treatment of a disease associated with abnormal levels of MDM2 protein; (2) And preparing a product for detecting MDM2 protein or diagnosing diseases related to the abnormal level of MDM2 protein.
  10. 10. The use according to claim 9, wherein the disease associated with abnormal MDM2 protein levels comprises malignant tumor, non-neoplastic disease, genetic disease or tissue damage caused by chemoradiotherapy; the malignant tumor comprises soft tissue sarcoma, glioblastoma, gastrointestinal stromal tumor, lung cancer, bladder cancer, lung cancer, colorectal cancer, breast cancer or prostate cancer; The non-neoplastic disease includes atherosclerosis, human papilloma virus infection, alzheimer's disease, parkinson's disease, huntington's disease, cerebral ischemia, or stroke; The hereditary diseases comprise Li-Fraomeni syndrome, hereditary breast cancer, hereditary ovarian cancer syndrome or familial adenomatous polyposis; the tissue injury caused by radiotherapy and chemotherapy comprises bone marrow injury or gastrointestinal mucosa injury.

Description

Anti-MDM 2 single-chain antibody and application thereof Technical Field The invention relates to the field of bioengineering, in particular to an anti-MDM 2 single-chain antibody and application thereof. Background MDM2 (mouse double-micro homolog 2) acts as an intracellular key E3 ubiquitin ligase, playing a central role in the negative feedback regulation of the p53 signal pathway. Under the condition of DNA damage stress, kinases such as ATM, CHK2 and the like are activated, the specific site of MDM2 is directly phosphorylated, the conformation or protein interaction capacity of the specific site is changed, and the combination of MDM2 and p53 is weakened, so that p53 is stabilized and the transcription function of the specific site is activated. This regulatory mechanism ensures that cells can rapidly initiate p 53-mediated DNA repair or apoptosis procedures in the face of genotoxic threats. In addition to direct regulation of p53, MDM2 is also able to form homo/heterodimers with MDMX, significantly enhancing the inhibition effect on p 53. Amplification of the MDM2 gene or overexpression of proteins is a common mechanism of tumorigenesis. It is estimated that about 7-10% of tumors have MDM2 gene amplification, and in soft tissue sarcomas, osteosarcomas, glioblastomas, etc., this proportion can be as high as 20-30%. MDM2 has attracted considerable attention as an important target for cancer treatment. Currently, hundreds of small molecule inhibitors have been evaluated in preclinical studies and many molecules tested in clinical trials, but no FDA approved MDM2 inhibitors are currently on the market. Single-chain antibody (ScFv) is an important outcome in the development of engineering antibody technology. The complete antigen binding unit is constructed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody end to end by means of a flexible short peptide linker with a length of 15-20 amino acids through exquisite molecular design. The design skillfully simulates an antigen binding pocket formed by non-covalent interaction of VH and VL in a natural antibody, so that the antigen binding pocket can completely retain the specific recognition capability on the antigen. Based on the characteristic of small molecular weight, scFv shows excellent tissue penetrating capability, can more effectively penetrate through the vascular wall with abnormal structure and high osmotic pressure in solid tumor and compact tumor matrix, and reaches a deep focus area which is difficult to effectively enrich by the traditional complete antibody. In addition, the smaller size of ScFv also results in faster blood clearance in vivo and shorter half-life. Meanwhile, from the aspect of molecular construction, the coding gene of the ScFv has simple structure, is easy to operate and modify, can be fused with various functional effector molecules through a mature genetic engineering technology, and is constructed into a multifunctional targeting fusion protein, thus providing a wide technical platform for developing a new-generation high-efficiency and accurate biological targeting medicament. MDM2 functions through a large and flat protein-protein interaction (PPI) interface, small molecule inhibitors are often difficult to completely destroy or effectively mimic these complex PPIs, and are prone to drug resistance due to binding site mutations. The development of a novel MDM2 ScFV targeting module is not only hopeful to break through the limitation of small molecule ligands, but also has great potential in the directions of MDM 2E 3 ligase function application development, tumor imaging, protein targeting drug development and the like. Disclosure of Invention The invention aims to provide a single-chain antibody of MDM2 protein developed by rabbit monoclonal antibody preparation technology and a screening method thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme: The first aspect of the present invention provides an antibody or antigen-binding fragment thereof which specifically binds to MDM2 protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising 3 CDRs, H-CDR1, H-CDR2 and H-CDR3 and a light chain variable region comprising 3 CDRs, L-CDR1, L-CDR2 and L-CDR3, the amino acid sequences of said H-CDR1, H-CDR2 and H-CDR3 being as follows Any one of the CDR1, CDR2 and CDR3 of the heavy chain variable region, wherein the amino acid sequences of the L-CDR1, L-CDR2 and L-CDR3 are as followsCDR1, CDR2, CDR3 of any one of the light chain variable regions shown. In some embodiments, the CDRs of the light chain variable region and the heavy chain variable region are selected from any one of the following: (a) The amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region shown as SEQ ID No. 7, and the amino acid sequences of